RESUMO
Fine particulate matter (PM2.5) can damage airway epithelial barriers. The anion transport system plays a crucial role in airway epithelial barriers. However, the detrimental effect and mechanism of PM2.5 on the anion transport system are still unclear. In this study, airway epithelial cells and ovalbumin (OVA)-induced asthmatic mice were used. In transwell model, the adenosine triphosphate (ATP)-induced transepithelial anion short-circuit current (Isc) and airway surface liquid (ASL) significantly decreased after PM2.5 exposure. In addition, PM2.5 exposure decreased the expression levels of P2Y2R, CFTR and cytoplasmic free-calcium, but ATP can increase the expressions of these proteins. PM2.5 exposure increased the levels of Th2-related cytokines of bronchoalveolar lavage fluid, lung inflammation, collagen deposition and hyperplasisa of goblet cells. Interestingly, the administration of ATP showed an inhibitory effect on lung inflammation induced by PM2.5. Together, our study reveals that PM2.5 impairs the ATP-induced transepithelial anion Isc through downregulating P2Y2R/CFTR pathway, and this process may participate in aggravating airway hyperresponsiveness and airway inflammation. These findings may provide important insights on PM2.5-mediated airway epithelial injury.
Assuntos
Asma , Regulador de Condutância Transmembrana em Fibrose Cística , Material Particulado , Receptores Purinérgicos P2Y2 , Animais , Camundongos , Receptores Purinérgicos P2Y2/metabolismo , Receptores Purinérgicos P2Y2/genética , Asma/metabolismo , Asma/patologia , Asma/tratamento farmacológico , Asma/induzido quimicamente , Asma/imunologia , Material Particulado/efeitos adversos , Material Particulado/toxicidade , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Trifosfato de Adenosina/metabolismo , Ovalbumina/imunologia , Transdução de Sinais/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação para Baixo/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/patologia , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologiaRESUMO
Recent data indicate that extracellular ATP affects wound healing efficacy via P2Y2-dependent signaling pathway. In the current work, we propose double-modified ATP analogue-alpha-thio-beta,gamma-methylene-ATP as a potential therapeutic agent for a skin regeneration. For the better understanding of structure-activity relationship, beside tested ATP analogues, the appropriate single-modified derivatives of target compound, such as alpha-thio-ATP and beta,gamma-methylene-ATP, were also tested in the context of their involvement in the activation of ATP-dependent purinergic signaling pathway via the P2Y2 receptor. The diastereomerically pure alpha-thio-modified-ATP derivatives were obtained using the oxathiaphospholane method as separate SP and RP diastereomers. Both the single- and double- modified ATP analogues were then tested for their impact on the viability and migration of human keratinocytes. The involvement of P2Y2-dependent purinergic signaling was analyzed in silico by molecular docking of the tested compounds to the P2Y2 receptor and experimentally by studying intracellular calcium mobilization in the human keratinocytes HaCaT. The effects obtained for ATP analogues were compared with the results for ATP as a natural P2Y2 agonist. To confirm the contribution of the P2Y2 receptor to the observed effects, the tests were also performed in the presence of the selective P2Y2 antagonist-AR-C118925XX. The ability of the alpha-thio-beta,gamma-methylene-ATP to influence cell migration was analyzed in vitro on the model HaCaT and MDA-MB-231 cells by wound healing assay and transwell migration test as well as in vivo using zebrafish system. The impact on tissue regeneration was estimated based on the regrowth rate of cut zebrafish tails. The in vitro and in vivo studies have shown that the SP-alpha-thio-beta,gamma-methylene-ATP analogue promotes regeneration-related processes, making it a suitable agent for enhance wound healing. Performed studies indicated its impact on the cell migration, induction of epithelial-mesenchymal transition and intracellular calcium mobilization. The enhanced regeneration of cut zebrafish tails confirmed the pro-regenerative activity of this ATP analogue. Based on the performed studies, the SP-alpha-thio-beta,gamma-methylene-ATP is proposed as a potential therapeutic agent for wound healing and skin regeneration treatment.
Assuntos
Trifosfato de Adenosina , Queratinócitos , Cicatrização , Peixe-Zebra , Cicatrização/efeitos dos fármacos , Humanos , Trifosfato de Adenosina/metabolismo , Animais , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Simulação de Acoplamento Molecular , Movimento Celular/efeitos dos fármacos , Receptores Purinérgicos P2Y2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Cálcio/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
BACKGROUND AND AIMS: Vascular smooth muscle cell (VSMC) dedifferentiation contributes substantively to vascular disease. VSMCs spontaneously release low levels of ATP that modulate vessel contractility, but it is unclear if autocrine ATP signaling in VSMCs is critical to the maintenance of the VSMC contractile phenotype. METHODS: We used pharmacological inhibitors to block ATP release in human aortic smooth muscle cells (HASMCs) for studying changes in VSMC differentiation marker gene expression. We employed RNA interference and generated mice with SMC-specific inducible deletion of the P2Y2 receptor (P2Y2R) gene to evaluate resulting phenotypic alterations. RESULTS: HASMCs constitutively release low levels of ATP that when blocked results in a significant decrease in VSMC differentiation marker gene expression, including smooth muscle actin (SMA), smooth muscle myosin heavy chain (SMMHC), SM-22α and calponin. Basal release of ATP represses transcriptional activation of the Krüppel-Like Factor 4 (KFL4) thereby preventing platelet-derived growth factor-BB (PDGF-BB) from inhibiting expression of SMC contractile phenotype markers. SMC-restricted conditional deletion of P2Y2R evoked dedifferentiation characterized by decreases in aortic contractility and contractile phenotype markers expression. This loss was accompanied by a transition to the synthetic phenotype with the acquisition of extracellular matrix (ECM) proteins characteristic of dedifferentiation, such as osteopontin and vimentin. CONCLUSIONS: Our data establish the first direct evidence that an autocrine ATP release mechanism maintains SMC cytoskeletal protein expression by inhibiting VSMCs from transitioning to a synthetic phenotype, and further demonstrate that activation of the P2Y2R by basally released ATP is required for maintenance of the differentiated VSMC phenotype.
Assuntos
Trifosfato de Adenosina , Becaplermina , Músculo Liso Vascular , Miócitos de Músculo Liso , Fenótipo , Receptores Purinérgicos P2Y2 , Animais , Receptores Purinérgicos P2Y2/metabolismo , Receptores Purinérgicos P2Y2/genética , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Humanos , Trifosfato de Adenosina/metabolismo , Camundongos , Becaplermina/metabolismo , Becaplermina/farmacologia , Células Cultivadas , Diferenciação Celular , Transdução de Sinais , Proteínas Proto-Oncogênicas c-sis/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Actinas/metabolismo , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Calponinas , Camundongos Knockout , Aorta/metabolismo , Aorta/citologia , Interferência de RNA , Desdiferenciação Celular , Cadeias Pesadas de Miosina/metabolismo , Cadeias Pesadas de Miosina/genética , Comunicação AutócrinaRESUMO
AIMS: Acute kidney injury (AKI) is a public health problem and represents a risk factor for cardiovascular diseases (CVD) and vascular damage. This study aimed to investigate the impact of AKI on purinergic components in mice aorta. MAIN METHODS: The kidney ischemia was achieved by the occlusion of the left kidney pedicle for 60 min, followed by reperfusion for 8 (IR8) and 15 (IR15) days. Renal function was assessed through biochemical assays, while gene expression levels were evaluated by RT-qPCR. KEY FINDINGS: Analyses of renal parameters showed renal remodeling through mass loss in the left kidney and hypertrophy of the right kidney in the IR15 group. Furthermore, after 15 days, local inflammation was evidenced in the aorta. Moreover, the aorta purinergic components were significantly impacted by the renal ischemia and reperfusion model, with increases in gene expression of the pro-inflammatory purinoceptors P2Y1, P2Y2, P2Y6, and P2X4, potentially contributing to the vessel inflammation. The expression of NTPDase2 and ecto-5'-nucleotidase were also significantly increased in the aorta of the same group. In addition, both ATP and AMP hydrolysis were significantly increased in the aorta from IR15 animals, driving the entire purinergic cascade to the production of the anti-inflammatory adenosine. SIGNIFICANCE: In short, this is the first time that inflammation of the aorta due to AKI was shown to have an impact on purinergic signaling components, with emphasis on the adenosinergic pathway. This seems to be closely implicated in the establishment of vascular inflammation in this model of AKI and deserves to be further investigated.
Assuntos
Injúria Renal Aguda , Rim , Traumatismo por Reperfusão , Transdução de Sinais , Animais , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Camundongos , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Injúria Renal Aguda/etiologia , Rim/metabolismo , Rim/irrigação sanguínea , Rim/patologia , Masculino , Aorta/metabolismo , Aorta/patologia , 5'-Nucleotidase/metabolismo , 5'-Nucleotidase/genética , Camundongos Endogâmicos C57BL , Receptores Purinérgicos/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Receptores Purinérgicos P2Y2/genéticaRESUMO
PURPOSE: The aim of the study was to evaluate the effect of silver nanoparticles hydrocolloids (AgNPs) on human corneal epithelial cells. Epithelial cells form the outermost and the most vulnerable to environmental stimuli layer of the cornea in the eye. Mechanical stress, UV radiation, and pathogens such as bacteria, viruses, and parasites challenge the fragile homeostasis of the eye. To help combat stress, infection, and inflammation wide portfolio of interventions is available. One of the oldest treatments is colloidal silver. Silver nanoparticle suspension in water is known for its anti-bacterial anti-viral and antiprotozoal action. However, AgNPs interact also with host cells, and the character of the interplay between corneal cells and silver seeks investigation. METHODS: The human epithelial corneal cell line (HCE-2) was cultured in vitro, treated with AgNPs, and subjected to UV. The cell's viability, migration, calcium concentration, and expression/protein level of selected proteins were investigated by appropriate methods including cytotoxicity tests, "wound healing" assay, Fluo8/Fura2 AM staining, qRT-PCR, and western blot. RESULTS: Incubation of human corneal cells (HCE-2) with AgNP did not affect cells viability but limited cells migration and resulted in altered calcium homeostasis, decreased the presence of ATP-activated P2X7, P2Y2 receptors, and enhanced the expression of PACAP. Furthermore, AgNPs pretreatment helped restrain some of the deleterious effects of UV irradiation. Interestingly, AgNPs had no impact on the protein level of ACE2, which is important in light of potential SARS-CoV-2 entrance through the cornea. CONCLUSIONS: Silver nanoparticles are safe for corneal epithelial cells in vitro.
Assuntos
Nanopartículas Metálicas , Prata , Humanos , Prata/metabolismo , Cálcio/metabolismo , Nanopartículas Metálicas/toxicidade , Receptores Purinérgicos P2Y2/metabolismo , Córnea , Células EpiteliaisRESUMO
The current study aimed to investigate the hypothesis that purinergic receptors P2Y1 and P2Y2 play a regulatory role in gene expression in unloaded muscle. ATP is released from cells through pannexin channels, and it interacts with P2Y1 and P2Y2 receptors, leading to the activation of markers of protein catabolism and a reduction in protein synthesis. To test this hypothesis thirty-two rats were randomly divided into four groups (8 per group): a non-treated control group (C), a group subjected to three days of hindlimb unloading with a placebo (HS), a group subjected to three days of hindlimb unloading treated with a P2Y1 receptor inhibitor, MRS2179 (HSM), and a group subjected to three days of hindlimb unloading treated with a P2Y2 receptor inhibitor, AR-C 118925XX (HSA). This study revealed several key findings following three days of soleus muscle unloading: 1: Inhibition of P2Y1 or P2Y2 receptors prevented the accumulation of ATP, the increase in IP3 receptor content, and the decrease in the phosphorylation of GSK-3beta. This inhibition also mitigated the reduction in the rate of protein synthesis. However, it had no significant effect on the markers of mTORC1-dependent signaling. 2: Blocking P2Y1 receptors prevented the unloading-induced upregulation of phosphorylated p38MAPK and partially reduced the increase in MuRF1mRNA expression. 3: Blocking P2Y2 receptors prevented muscle atrophy during unloading, partially maintained the levels of phosphorylated ERK1/2, reduced the increase in mRNA expression of MAFbx, ubiquitin, and IL-6 receptor, prevented the decrease in phosphorylated AMPK, and attenuated the increase in phosphorylated p70S6K. Taken together, these results suggest that the prevention of muscle atrophy during unloading, as achieved by the P2Y2 receptor inhibitor, is likely mediated through a reduction in catabolic processes and maintenance of energy homeostasis. In contrast, the P2Y1 receptor appears to play a relatively minor role in muscle atrophy during unloading.
Assuntos
Músculo Esquelético , Transdução de Sinais , Animais , Ratos , Trifosfato de Adenosina/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Receptores Purinérgicos P2Y1/metabolismo , Receptores Purinérgicos P2Y2/genética , Receptores Purinérgicos P2Y2/metabolismoRESUMO
Several P2Y nucleotide receptors have been shown to be involved in the early stage of adipocyte differentiation in vitro and insulin resistance in obese mice; however, the exact receptor subtype(s) and its underlying molecular mechanism in relevant human cells are unclear. Here, using human primary visceral preadipocytes as a model, we found that during preadipocyte-to-mature adipocyte differentiation, the P2Y2 nucleotide receptor (P2Y2R) was the most upregulated subtype among the eight known P2Y receptors and the only one further dramatically upregulated after inflammatory TNFα treatment. Functional studies indicated that the P2Y2R induced intracellular Ca2+, ERK1/2, and JNK signaling but not the p38 pathway. In addition, stimulation of the P2Y2R suppressed basal and insulin-induced phosphorylation of AKT, accompanied by decreased GLUT4 membrane translocation and glucose uptake in mature adipocytes, suggesting a role of P2Y2R in insulin resistance. Mechanistically, we found that activation of P2Y2R did not increase lipolysis but suppressed PIP3 generation. Interestingly, activation of P2Y2R triggered Gi-protein coupling, and pertussis toxin pretreatment largely inhibited P2Y2R-mediated ERK1/2 signaling and cAMP suppression. Further, treatment of the cells with AR-C 118925XX, a selective P2Y2R antagonist, significantly inhibited adipogenesis, and P2Y2R knockout decreased mouse body weight gain with smaller eWAT mass infiltrated with fewer macrophages as compared to WT mice in response to a Western diet. Thus, we revealed that terminal adipocyte differentiation and inflammation selectively upregulate P2Y2R expression and that P2Y2R mediates insulin resistance by suppressing the AKT signaling pathway, highlighting P2Y2R as a potential new drug target to combat obesity and type-2 diabetes.
Assuntos
Adipogenia , Resistência à Insulina , Receptores Purinérgicos P2Y2 , Animais , Humanos , Camundongos , Adipócitos/citologia , Adipócitos/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Resistência à Insulina/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Purinérgicos P2Y2/genética , Receptores Purinérgicos P2Y2/metabolismo , Transdução de Sinais/genética , Células Cultivadas , Camundongos Endogâmicos C57BL , Regulação para Cima , Transportador de Glucose Tipo 4/metabolismo , Transporte Proteico/genética , Lipólise/genética , Adipogenia/genéticaRESUMO
Allergic airway inflammation (AAI) is a chronic respiratory disease that is considered a severe restriction in daily life and is accompanied by a constant risk of acute aggravation. It is characterized by IgE-dependent activation of mast cells, infiltration of eosinophils, and activated T-helper cell type 2 (Th2) lymphocytes into airway mucosa. Purinergic receptor signaling is known to play a crucial role in inducing and maintaining allergic airway inflammation. Previous studies in an ovalbumin (OVA)-alum mouse model demonstrated a contribution of the P2Y2 purinergic receptor subtype (P2RY2) in allergic airway inflammation. However, conflicting data concerning the mechanism by which P2RY2 triggers AAI has been reported. Thus, we aimed at elucidating the cell-type-specific role of P2RY2 signaling in house dust mite (HDM)-driven model of allergic airway inflammation. Thereupon, HDM-driven AAI was induced in conditional knockout mice, deficient or intact for P2ry2 in either alveolar epithelial cells, hematopoietic cells, myeloid cells, helper T cells, or dendritic cells. To analyze the functional role of P2RY2 in these mice models, flow cytometry of bronchoalveolar lavage fluid (BALF), cytokine measurement of BALF, invasive lung function measurement, HDM re-stimulation of mediastinal lymph node (MLN) cells, and lung histology were performed. Mice that were subjected to an HDM-based model of allergic airway inflammation resulted in reduced signs of acute airway inflammation including eosinophilia in BALF, peribronchial inflammation, Th2 cytokine production, and bronchial hyperresponsiveness in mice deficient for P2ry2 in alveolar epithelial cells, hematopoietic cells, myeloid cells, or dendritic cells. Furthermore, the migration of bone-marrow-derived dendritic cells and bone-marrow-derived monocytes, both deficient in P2ry2, towards ATP was impaired. Additionally, we found reduced levels of MCP-1/CCL2 and IL-8 homologues in the BALF of mice deficient in P2ry2 in myeloid cells and lower concentrations of IL-33 in the lung tissue of mice deficient in P2ry2 in alveolar epithelial cells. In summary, our results show that P2RY2 contributes to HDM-induced airway inflammation by mediating proinflammatory cytokine production in airway epithelial cells, monocytes, and dendritic cells and drives the recruitment of lung dendritic cells and monocytes.
Assuntos
Citocinas , Pulmão , Camundongos , Animais , Receptores Purinérgicos P2Y2/genética , Receptores Purinérgicos P2Y2/metabolismo , Citocinas/metabolismo , Pulmão/patologia , Pyroglyphidae , Inflamação/metabolismoRESUMO
Lung ischemia-reperfusion injury (IRI), characterized by inflammation, vascular permeability, and lung edema, is the major cause of primary graft dysfunction after lung transplantation. Here, we investigated the cellular mechanisms underlying lung IR-induced activation of endothelial TRPV4 channels, which play a central role in lung edema and dysfunction after IR. In a left lung hilar-ligation model of IRI in mice, we found that lung IRI increased the efflux of ATP through pannexin 1 (Panx1) channels at the endothelial cell (EC) membrane. Elevated extracellular ATP activated Ca2+ influx through endothelial TRPV4 channels downstream of purinergic P2Y2 receptor (P2Y2R) signaling. P2Y2R-dependent activation of TRPV4 channels was also observed in human and mouse pulmonary microvascular endothelium in ex vivo and in vitro models of IR. Endothelium-specific deletion of P2Y2R, TRPV4, or Panx1 in mice substantially prevented lung IRI-induced activation of endothelial TRPV4 channels and lung edema, inflammation, and dysfunction. These results identify endothelial P2Y2R as a mediator of the pathological sequelae of IRI in the lung and show that disruption of the endothelial Panx1-P2Y2R-TRPV4 signaling pathway could be a promising therapeutic strategy for preventing lung IRI after transplantation.
Assuntos
Traumatismo por Reperfusão , Canais de Cátion TRPV , Humanos , Animais , Camundongos , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Receptores Purinérgicos P2Y2/genética , Receptores Purinérgicos P2Y2/metabolismo , Pulmão/metabolismo , Traumatismo por Reperfusão/metabolismo , Células Endoteliais/metabolismo , Inflamação/metabolismo , Trifosfato de Adenosina/metabolismo , Edema/metabolismo , Edema/patologia , Proteínas do Tecido Nervoso/metabolismo , Conexinas/genética , Conexinas/metabolismoRESUMO
Serratia marcescens is an opportunistic human pathogen involved in antibiotic-resistant hospital acquired infections. Upon contact with the host epithelial cell and prior to internalization, Serratia induces an early autophagic response that is entirely dependent on the ShlA toxin. Once Serratia invades the eukaryotic cell and multiples inside an intracellular vacuole, ShlA expression also promotes an exocytic event that allows bacterial egress from the host cell without compromising its integrity. Several toxins, including ShlA, were shown to induce ATP efflux from eukaryotic cells. Here, we demonstrate that ShlA triggered a nonlytic release of ATP from Chinese hamster ovary (CHO) cells. Enzymatic removal of accumulated extracellular ATP (eATP) or pharmacological blockage of the eATP-P2Y2 purinergic receptor inhibited the ShlA-promoted autophagic response in CHO cells. Despite the intrinsic ecto-ATPase activity of CHO cells, the effective concentration and kinetic profile of eATP was consistent with the established affinity of the P2Y2 receptor and the known kinetics of autophagy induction. Moreover, eATP removal or P2Y2 receptor inhibition also suppressed the ShlA-induced exocytic expulsion of the bacteria from the host cell. Blocking α5ß1 integrin highly inhibited ShlA-dependent autophagy, a result consistent with α5ß1 transactivation by the P2Y2 receptor. In sum, eATP operates as the key signaling molecule that allows the eukaryotic cell to detect the challenge imposed by the contact with the ShlA toxin. Stimulation of P2Y2-dependent pathways evokes the activation of a defensive response to counteract cell damage and promotes the nonlytic clearance of the pathogen from the infected cell.
Assuntos
Autofagia , Interações Hospedeiro-Patógeno , Integrina alfa5beta1 , Receptores Purinérgicos P2Y2 , Serratia , Toxinas Biológicas , Animais , Cricetinae , Trifosfato de Adenosina/metabolismo , Autofagia/efeitos dos fármacos , Células CHO , Cricetulus , Exocitose/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Integrina alfa5beta1/antagonistas & inibidores , Integrina alfa5beta1/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Serratia/química , Serratia/efeitos dos fármacos , Serratia/fisiologia , Toxinas Biológicas/farmacologia , HumanosRESUMO
We previously reported that radiotherapyresistant (RTR) triple negative breast cancer (TNBC) cells upregulate the expression of endothelialspecific molecule1 (ESM1) compared with TNBC cells. In addition, ESM1 is involved in an increased proliferation and invasion of RTRTNBC cells compared with TNBC cells. It was further identified that, in RTRTNBC cells, P2Y2 purinergic receptor (P2Y2R)mediated activation of p21activated kinase 1 (PAK1), protein kinase C (PKC), cJun Nterminal kinase (JNK) and p38 MAPKs is related to ESM1 expression via forkhead box O1 (FoxO1) regulation. Notably, it has been reported that P2Y2R mediates the transactivation of vascular epithelial growth factor receptor 2 (VEGFR2), and VEGFR2 is known to be involved in ESM1 expression. Therefore, in the present study, the involvement of VEGFR2 in the P2Y2Rmediated ESM1 upregulation in RTRTNBC cells and the relationship between P2Y2R and VEGFR2 activation was further examined. Western blotting and reverse transcriptionPCR were used to monitor the expression of ESM1, and the results demonstrated that extracellular ATP treatment regulated the expression of ESM1 in a P2Y2Rdependent manner in RTRMDAMB231 cells. In addition, extracellular ATP activated Src and VEGFR2 after 5 min of incubation, which was abolished by knockdown of P2Y2R expression. VEGFR2 activation in response to ATP was also decreased by inhibiting Src activity, suggesting that ATPactivated P2Y2R regulates VEGFR2 phosphorylation via Src activation. Furthermore, ATPinduced ESM1 expression was decreased by transfection with VEGFR2 small interfering RNA (siRNA). ESM1related signaling molecules, PAK1, PKC, JNK and p38 MAPKs, and the transcriptional regulator, FoxO1, which were activated by ATP, were also decreased following transfection with VEGFR2 siRNA. These results suggest that P2Y2Rmediated transactivation of VEGFR2 through Src phosphorylation is associated with ESM1 overexpression in RTRTNBC cells.
Assuntos
Receptores Purinérgicos P2Y2 , Neoplasias de Mama Triplo Negativas , Receptor 2 de Fatores de Crescimento do Endotélio Vascular , Humanos , Trifosfato de Adenosina/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Receptores de Fatores de Crescimento/metabolismo , RNA Interferente Pequeno/metabolismo , Ativação Transcricional , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/radioterapia , Receptores Purinérgicos P2Y2/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Pancreatic ductal adenocarcinoma (PDAC) continues to show no improvement in survival rates. One aspect of PDAC is elevated ATP levels, pointing to the purinergic axis as a potential attractive therapeutic target. Mediated in part by highly druggable extracellular proteins, this axis plays essential roles in fibrosis, inflammation response, and immune function. Analyzing the main members of the PDAC extracellular purinome using publicly available databases discerned which members may impact patient survival. P2RY2 presents as the purinergic gene with the strongest association with hypoxia, the highest cancer cell-specific expression, and the strongest impact on overall survival. Invasion assays using a 3D spheroid model revealed P2Y2 to be critical in facilitating invasion driven by extracellular ATP. Using genetic modification and pharmacological strategies, we demonstrate mechanistically that this ATP-driven invasion requires direct protein-protein interactions between P2Y2 and αV integrins. DNA-PAINT super-resolution fluorescence microscopy reveals that P2Y2 regulates the amount and distribution of integrin αV in the plasma membrane. Moreover, receptor-integrin interactions were required for effective downstream signaling, leading to cancer cell invasion. This work elucidates a novel GPCR-integrin interaction in cancer invasion, highlighting its potential for therapeutic targeting.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Linhagem Celular Tumoral , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/patologia , Invasividade Neoplásica/genética , Trifosfato de Adenosina/metabolismo , Integrinas/metabolismo , Proliferação de Células/genética , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Receptores Purinérgicos P2Y2/genética , Receptores Purinérgicos P2Y2/metabolismoRESUMO
Muscle regeneration is indispensable for skeletal muscle health and daily life when injury, muscular disease, and aging occur. Among the muscle regeneration, muscle stem cells' (MuSCs) activation, proliferation, and differentiation play a key role in muscle regeneration. Purines bind to its specific receptors during muscle development, which transmit environmental stimuli and play a crucial role of modulator of muscle regeneration. Evidences proved P2R expression during development and regeneration of skeletal muscle, both in human and mouse. In contrast to P2XR, which have been extensively investigated in skeletal muscles, the knowledge of P2YR in this tissue is less comprehensive. This review summarized muscle regeneration via P2Y1R and P2Y2R and speculated that P2Y1R and P2Y2R might be potential molecular triggers for MuSCs' activation and proliferation via the p-ERK1/2 and PLC pathways, explored their cascade effects on skeletal muscle, and proposed P2Y1/2 receptors as potential pharmacological targets in muscle regeneration, to advance the purinergic signaling within muscle and provide promising strategies for alleviating muscular disease.
Assuntos
Músculo Esquelético , Doenças Musculares , Animais , Humanos , Camundongos , Diferenciação Celular , Músculo Esquelético/metabolismo , Doenças Musculares/metabolismo , Regeneração/fisiologia , Transdução de Sinais , Receptores Purinérgicos P2Y1/metabolismo , Receptores Purinérgicos P2Y2/metabolismoRESUMO
Selinexor is a first-in-class inhibitor of the nuclear exportin XPO1 that was recently approved by the US Food and Drug Administration for the treatment of multiple myeloma and diffuse large B-cell lymphoma. In relapsed/refractory acute myeloid leukemia (AML), selinexor has shown promising activity, suggesting that selinexor-based combination therapies may have clinical potential. Here, motivated by the hypothesis that selinexor's nuclear sequestration of diverse substrates imposes pleiotropic fitness effects on AML cells, we systematically catalog the pro- and anti-fitness consequences of selinexor treatment. We discover that selinexor activates PI3Kγ-dependent AKT signaling in AML by upregulating the purinergic receptor P2RY2. Inhibiting this axis potentiates the anti-leukemic effects of selinexor in AML cell lines, patient-derived primary cultures and multiple mouse models of AML. In a syngeneic, MLL-AF9-driven mouse model of AML, treatment with selinexor and ipatasertib outperforms both standard-of-care chemotherapy and chemotherapy with selinexor. Together, these findings establish drug-induced P2RY2-AKT signaling as an actionable consequence of XPO1 inhibition in AML.
Assuntos
Leucemia Mieloide Aguda , Proteínas Proto-Oncogênicas c-akt , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Carioferinas/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Estados Unidos , Proteína Exportina 1RESUMO
P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular nucleotides. The P2Y2 receptor subtype is expressed in a variety of cell types and plays an important role in physiological and pathophysiological processes such as inflammatory responses and neuropathic pain. Based on this, the P2Y2 has been identified as an important drug target. The specificity of current P2Y2 receptor modulators is relatively poor, and currently, specific and efficient P2Y2 receptor modulators and efficient screening strategies are lacking. In this study, a cell model based on calcium-activated chloride channels (CaCCs) was established that can detect changes in intracellular calcium concentrations and can be used to high-throughput screen for P2Y2 receptor-specific regulators. This screening strategy is suitable for screening of most G-protein-coupled receptor regulators that mediate increases in intracellular calcium signals. The cell model consists of three components that include the endogenously expressed P2Y2 receptor protein, the exogenously expressed calcium-activated chloride channel Anoctamin-1 (Ano1), and a yellow fluorescent protein mutant expressed within the cell that is highly sensitive to iodine ions. This model will allow for high-throughput screening of GPCR regulators that mediate increased intracellular calcium signaling using the calcium-activated transport of iodide ions by Ano1. We verified the ability of the model to detect intracellular calcium ion concentration using fluorescence quenching kinetic experiments by applying existing P2Y2 agonists and inhibitors to validate the screening function of the model, and we also evaluated the performance of the model in the context of high-throughput screening studies. The experimental results revealed that the model could sensitively detect intracellular calcium ion concentration changes and that the model was accurate in regard to detecting P2Y2 modulators. The resultant value of the Z-factor was 0.69, thus indicating that the model possesses good sensitivity and specificity.
Assuntos
Cálcio , Ensaios de Triagem em Larga Escala , Cálcio/metabolismo , Sinalização do Cálcio , Nucleotídeos/metabolismo , Receptores Purinérgicos P2Y2/genética , Receptores Purinérgicos P2Y2/metabolismoRESUMO
Purpose: It is well known that inflammation plays a key role in complex pathological progressions of alcohol-associated liver disease (ALD). To date, effective therapy for ALD is lacking. P2Y2 receptor (P2Y2R), a G protein-coupled P2Y purinergic receptor, represents a novel pharmacological target in many inflammations. Methods: The alcohol-associated liver injury and inflammation mouse model was established. The effect of P2Y2R on alcohol-induced liver injury and inflammation was evaluated using quantitative real-time PCR, Western blot and immunohistochemical assay. An alcohol-stimulated (100 mmol/L, for 24 h) AML-12 cell model was established. Different agonists, antagonists and P2Y2R siRNA were used to explore the possible mechanisms of P2Y2R. Results: In vivo, results showed that the hepatoprotective effect of P2Y2R blockade by significantly suppressed liver structural abnormalities and lipid infiltration, and decreased levels of ALT/AST and TNF-α/IL-1ß in the high dosage group of suramin (20 mg/kg) compared to control diet (CD)-fed mice. At the same time, we found that alcohol feeding promoted the phosphorylation of EGFR and ERK1/2, both of which were effectively inhibited by suramin (20 mg/kg). In vitro, suramin or P2Y2R silencing effectively inhibited the phosphorylation of EGFR and ERK1/2, similar to the down-regulated effects of their corresponding inhibitors (EGFR inhibitor AG1478 and ERK1/2 inhibitor U0126) accompanied by reduced levels of TNF-α and IL-1ß compared to alcohol-induced AML-12 cell. In addition, we found that silencing P2Y2R attenuated the apoptosis of hepatocyte. Conclusion: Our findings suggest that P2Y2R regulates alcoholic liver inflammation by targeting the EGFR-ERK1/2 signaling pathway and plays an important role in hepatocyte apoptosis, which may provide new ideas for the development of methods to treat ALD.
Assuntos
Leucemia Mieloide Aguda , Sistema de Sinalização das MAP Quinases , Animais , Receptores ErbB , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Leucemia Mieloide Aguda/metabolismo , Fígado/metabolismo , Camundongos , Receptores Purinérgicos P2Y2/metabolismo , Transdução de Sinais , Suramina/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: The G protein-coupled receptor P2RY2 protein of the purinergic receptor family is involved in the pathogenesis of Alzheimer's disease (AD). Naturally occurring antibodies against P2RY2 (NAbs-P2RY2) are present in human plasma, with their clinical relevance in AD unknown. OBJECTIVE: To explore the alteration of NAbs-P2RY2 in AD patients and its associations with biomarkers and cognition of AD patients. METHODS: The levels of naturally occurring antibodies against the four extracellular domains of P2RY2 (NAbs-P2RY2-1, NAbs-P2RY2-2, NAbs-P2RY2-3, and NAbs-P2RY2-4) were measured in the plasma of 55 AD patients, 28 non-AD dementia patients, and 70 cognitively normal participants. The correlations of autoantibody levels with cognitive scale scores, AD plasma biomarkers, and brain amyloid burden were examined. RESULTS: NAbs-P2RY2-1, NAbs-P2RY2-3, and NAbs-P2RY2-4 were reduced in AD patients. Plasma levels of NAbs-P2RY2-2 and NAbs-P2RY2-3 levels were positively associated with cognitive and functional performances. Among these antibodies, plasma NAbs-P2RY2-2 levels were positively associated with plasma amyloid-ß 42 levels. While plasma NAbs-P2RY2-3 levels were negatively associated with brain amyloid burden in AD patients. CONCLUSION: These findings indicate an alteration of humoral immunity against P2RY2 in AD patients. Further mechanistical investigations are needed to reveal the role of NAbs-P2RY2 in the pathogenesis of AD.
Assuntos
Doença de Alzheimer , Amiloidose , Disfunção Cognitiva , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Amiloidose/metabolismo , Autoanticorpos , Biomarcadores , Encéfalo/patologia , Disfunção Cognitiva/metabolismo , Humanos , Receptores Purinérgicos P2Y2/metabolismo , Proteínas tau/metabolismoRESUMO
The aim of this study was to investigate the effect of the P2Y2 receptor (P2Y2R) signaling pathway on neuronal regeneration and angiogenesis during spinal cord injury (SCI). The rats were randomly divided into 3 groups, including the sham+dimethyl sulfoxide (DMSO), SCI+DMSO, and SCI+P2Y2R groups. The SCI animal models were constructed. A locomotor rating scale was used for behavioral assessments. The apoptosis of spinal cord tissues was detected by TUNEL staining. The expression levels of P2Y2R, GFAP, nestin, Tuj1, and CD34 were detected by immunofluorescence staining, and the expression levels of TNF-α, IL-1ß, and IL-6 were detected by enzyme-linked immunosorbent assay. The locomotor score in the model group was significantly lower than the sham group. The expression of P2Y2R was increased after SCI. The expression levels of TNF-α, IL-1ß, and IL-6 were increased remarkably in the SCI model group compared with the sham group. The P2Y2R inhibitor relieved neuronal inflammation after SCI. Compared with the sham group, the apoptotic rate of spinal cord tissue cells in the model group was significantly increased. The P2Y2R inhibitor reduced the apoptosis of the spinal cord tissue. The expressions of CD34, Tuj1, and nestin in the model group were decreased, while the expressions of GFAP and P2Y2R were increased. The P2Y2R inhibitor reversed their expression levels. The P2Y2R inhibitor could alleviate SCI by relieving the neuronal inflammation, inhibiting the spinal cord tissue apoptosis, and promoting neuronal differentiation and vascular proliferation after SCI. P2Y2R may serve as a target for the treatment of SCI.
Assuntos
Regeneração Nervosa , Neurônios , Receptores Purinérgicos P2Y2 , Traumatismos da Medula Espinal , Medula Espinal , Animais , Modelos Animais de Doenças , Neovascularização Patológica , Regeneração Nervosa/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2Y2/metabolismo , Recuperação de Função Fisiológica , Medula Espinal/irrigação sanguínea , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologiaRESUMO
Mechanical stimulation of cultured keratinocytes and a living epidermis increases intracellular calcium ion concentrations ([Ca2+]i) in stimulated cells. This action propagates a Ca2+ wave to neighboring keratinocytes via ATP/P2Y2 receptors. Recent behavioral, pharmacological studies revealed that exogenous ATP induces itching via P2X3 receptors in mice. We previously showed that alloknesis occurs when an external stimulus is applied to the skin with increased epidermal histamine in the absence of spontaneous pruritus. Based on these results, we investigated the effects of histamine at a concentration that does not cause itching on ATP-induced itching. The mean number of scratching events induced by the mixture of ATP and histamine increased by 28% over the sum of that induced by histamine alone or ATP alone. A317491, a P2X3 receptor antagonist, suppressed the mixture-induced scratching more often than the ATP-induced scratching. Next, we examined the ATP-induced [Ca2+]i change before and after histamine stimulation using normal human epidermal keratinocytes. Some cells did not respond to ATP before histamine stimulation but responded to ATP afterward, the phenomenon suppressed by chlorpheniramine maleate. These findings suggest that histamine enhances ATP-induced itching and that a potential mechanism could involve increased responsiveness to ATP in keratinocytes.
Assuntos
Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Histamina/farmacologia , Queratinócitos/metabolismo , Prurido/induzido quimicamente , Prurido/tratamento farmacológico , Animais , Cálcio/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Masculino , Camundongos Endogâmicos ICR , Fenóis/farmacologia , Fenóis/uso terapêutico , Estimulação Física , Compostos Policíclicos/farmacologia , Compostos Policíclicos/uso terapêutico , Agonistas do Receptor Purinérgico P2X , Receptores Purinérgicos P2X3/metabolismo , Receptores Purinérgicos P2Y2/metabolismoRESUMO
Pannexin 1 (Panx1), an ATP-efflux pathway, has been linked with inflammation in pulmonary capillaries. However, the physiological roles of endothelial Panx1 in the pulmonary vasculature are unknown. Endothelial transient receptor potential vanilloid 4 (TRPV4) channels lower pulmonary artery (PA) contractility and exogenous ATP activates endothelial TRPV4 channels. We hypothesized that endothelial Panx1-ATP-TRPV4 channel signaling promotes vasodilation and lowers pulmonary arterial pressure (PAP). Endothelial, but not smooth muscle, knockout of Panx1 increased PA contractility and raised PAP in mice. Flow/shear stress increased ATP efflux through endothelial Panx1 in PAs. Panx1-effluxed extracellular ATP signaled through purinergic P2Y2 receptor (P2Y2R) to activate protein kinase Cα (PKCα), which in turn activated endothelial TRPV4 channels. Finally, caveolin-1 provided a signaling scaffold for endothelial Panx1, P2Y2R, PKCα, and TRPV4 channels in PAs, promoting their spatial proximity and enabling signaling interactions. These results indicate that endothelial Panx1-P2Y2R-TRPV4 channel signaling, facilitated by caveolin-1, reduces PA contractility and lowers PAP in mice.